Academic Sciences International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 4, Issue 1, 2012 Research Article IN VITRO CYTOTOXIC ACTIVITIES OF CALOTROPIS PROCERA LATEX AND FLOWER EXTRACTS AGAINST MCF-7 AND HeLa CELL LINE CULTURES PUSAPATI MADAN RANJIT*, KRISHNA PRIYA M, SILPA P, NAGALAKSHMI V, ANJALI M, GIRISH K, AND Y.A.CHOWDARY Department of Pharmaceutical Biotechnology, NRI College of Pharmacy, Pothavarappadu Village, Agiripalli Mandal, Krishna District, A.P, India. Email: madanranjit@gmail.com ABSTRACT Received: 12 Feb 2011, Revised and Accepted: 18 May 2011 Calotropis procera is a wild growing plant with multifarious medicinal properties. The present study was carried out to evaluate the effect of dried latex (DL) and flowers of C.procera and its ethanolic extracts against MCF-7 and HeLa cell line cultures. The MTT assay developed by Mosmann, it was modified and used to determine the inhibitory effects of test compounds on cell growth in vitro. Different concentrations of test compounds were used (Sample: 1 DL extract, Sample: 2 Flower extract) (4, 8, 16, 32, 64, 128, 256 and 512 µg/ml) in triplicates to achieve a final volume of 100 µl and then cultured for 48 hr. The standard (Tamoxifen) was prepared as 1.0 mg/ml concentration stock solutions in PBS. Culture medium and solvent were used as controls. The standard drug tamoxifen inhibits 60.46% breast cancer (MCF-7) cells when treated at 512 μg/ml concentration. At initial concentrations of the standard drug, the percent inhibition was reduced whereas both the samples: 1 and 2 displayed a dose-dependent inhibition at the tested concentrations. The ethanolic extract of DL and flowers showed cytotoxic properties against both MCF-7 and HeLa cells in a dose dependent manner. Keywords: Calotropis procera, MCF-7 cell line, HeLa cell line, In vitro cytotoxicity INTRODUCTION Calotropis procera (Ait.)R.Br. (Asclepiadaceae) commonly known, as Arka is a popular medicinal plant found throughout the tropics of Asia and Africa and is used in many traditional systems of medicine. Important factors of the various parts of this plant have been widely reported. In traditional medicinal system, it has been used for a variety of diseases condition that include leprosy, ulcers, tumors and piles 1.The plant has also been used as an emetic and purgative. Besides, it has been shown to exhibit spasmogenic and carminative properties 1, 2. The flowers and every part of this plant contain latex in abundance 3. Different parts of the Calotropis procera are used in treatment of various diseases in folk medicine, and their effects were confirmed by various scientific experiments 4. Besides, the chloroform extract of the roots has been shown to possess antiinflammatory and analgesic properties 5. The anti-inflammatory and analgesic properties were exhibited by the latex of the plant which is abundant in the aerial parts 6, 7. The latex also exhibits free radical scavenging and antioxidant properties 8, 9, in addition to its application as an antidote for snake poisoning 10. It also showed antitumor activity tested in trials using different tumor cell lines and it was able to inhibit the development of cell lines HL-60 (leukemia), HCT-8 (colon cancer), MDA-MB-435 (Breast cancer) and F295 (cell cancer of the brain) when analyzed 11. This study showed the in vitro cytotoxic activities of ethanolic extract of dry latex and flowers of Calotropis procera on MCF-7 and HeLa cell lines. MATERIALS AND METHODS Plant Material The flowers and latex were collected in month of September from-2010 out fields of Agiripalli Mandal, Krishna district, identified and herbarium specimen was deposited in the department of Pharmacognosy with specimen No.NRI/COL/P.COG/1/PF (Flowers) and NRI/COL/P.COG/2/PL (Latex). The latex was collected from the aerial parts of the plant growing in wild, it was air dried under shade at ambient temperature and ground to small granules of dried latex (DL). Preparation of extracts The shade-dried powder of flowers was subjected to extraction in soxhlet extractor with 70% EtOH for 70 hours (extract yield: 9%) and Dry latex (DL) was macerated with 70% EtOH and extract (extract yield: 4.5%) was collected. Both extracts were evaporated to dryness and stored at 4 C until used. In Vitro Cytotoxicity Studies Maintenance of Cell Lines Breast cancer (MCF-7) cells and carcinoma of cervix (HeLa) cells were maintained in Dulbecco s modified essential medium (DMEM) supplemented with 4.5 g/l glucose and 2 mm L-glutamine and 5% foetal bovine serum (FBS, Growth medium) at 37 C in 5% CO2 incubator. Cell culture Carcinoma of cervix (HeLa), breast, melanoma and thyroid cells were maintained in Dulbecco s modified essential medium (DMEM) supplemented with 4.5 g/l glucose, 2 mm L-glutamine and 5% fetal bovine serum (FBS, Growth medium) at 37 C in 5% CO2 incubator. MTT assay The MTT assay developed by Mosmann was modified and used to determine the inhibitory effects of test compounds on cell growth in vitro 12. In brief, the trypsinized cells from T-25 flask were seeded in each well of 96-well flat-bottomed tissue culture plate at a density of 5x10 3 cells well in growth medium and cultured at 37 C in 5% CO2 to adhere. After 48hr incubation, the supernatant was discarded and the cells pretreated with growth medium, subsequently mixed with different concentrations of test compounds (Sample: 1 DL extract, Sample: 2 Flower extract) (4, 8, 16, 32, 64, 128, 256 and 512 µg/ml) in triplicates to achieve a final volume of 100 µl and then cultured for 48 hr. The standard (Tamoxifen) was prepared as 1.0 mg/ml concentration stock solutions in PBS. Culture medium and solvent were used as controls. Each well then received 5 µl of fresh MTT (0.5mg/ml in PBS) followed by incubation for 2hr at 37 C. The supernatant growth medium was removed from the wells and replaced with 100 µl of DMSO to solubilize the colored formazan product. After 30 min incubation, the absorbance (OD) of the culture plate was read at a wavelength of 570 nm on an ELISA reader, Anthos 2020 spectrophotometer. RESULTS AND DISCUSSION Carcinoma of breast Results in table 1 showed that the standard drug tamoxifen inhibits 60.46% breast cancer (MCF-7) cells when treated at 512 μg/ml concentration. At initial concentrations of the standard drug, the
percent inhibition was reduced whereas both the samples: 1 and 2 displayed a dose-dependent inhibition (Table 2 and 3) at the tested concentrations. However, more than 50% inhibition was not observed in both the cases. Carcinoma of cervix It has been observed that the percentage inhibitory activity of the two samples 1 and 2 tested against carcinoma of cervix (HeLa cells) yielded much lower percentage inhibition than the standard drug tamoxifen. Results in table 4 showed that more than 50% inhibition was observed at 128 μg/ml concentration of the standard drug, whereas both the samples at highest tested dose displayed 41-43% inhibition only (Table 6 and 5). The present study was carried out to evaluate the in vitro cytotoxic activities of ethanolic extract of both DL and flower of C.procera on MCF-7 and HeLa cells. However, from the graphs (Fig. 1, Fig. 2) it was evident that a dose-dependent relationship was observed for all the samples under study. This shows that the samples have some growth inhibitory characteristics against carcinoma of breast cancer and cervix cell line (Fig. 3). Inhibitory data as well as comparative graphs are also provided. Smit et al reported that C. procera flower extract showed the potent in vitro cytotoxic activity, which is comparable to standard anticancer drug cisplatin 13. Another report explained that latex of C. procera exhibits cytotoxic properties like standard anticancer drugs podophyllotoxin and cyclophosphamide 14. The latex has also showed cytotoxicity corroborated by morphological alterations in leukemia cells, such as chromatin condensation, DNA fragmentation and cell volume reduction 15, 16. Another study reported that, X15-myc transgenic mice treated with DL (400mg/kg) for a period of 15 week, protected mice from malignant changes occurring in liver while sinusoidal architecture and cellular integrity were slightly disrupted as compared to normal and hydropic changes were observed. DL produced a significant decrease in the serum vascular endothelial growth factor (VEGF) levels of X15-myc transgenic mice. The methanolic extract (ME) of DL was induced cell death in cell lines viz., Huh-7 and COS-1 cells. It was evaluated by using tetrazolium (MTT) assay. The ME was subjected to silica gel G step column chromatography using a combination of non-polar and polar solvents and 11 fractions were obtained. Out of 11 fractions, fraction 8 exhibited potent cytotoxic effect on both the cell lines. However, a marginal effect on the killing of non-cancerous cell lines suggested a high degree of selectivity for transformed cells. Such differential killing of cancerous cells could relate to their altered metabolic status and/or membrane properties. The cytotoxic effect of DL was accompanied by intracellular fragmentation of target cell DNA 17. Our study reported that ethanolic extract of DL and flowers showed cytotoxic properties against both MCF-7 and HeLa cells in a dose dependent manner. As per our knowledge, our studies reported that both ethanolic extracts of DL and flower showed cytotoxic effects on estrogen positive cell line (i.e.mcf-7) and carcinoma of cervix (i.e. HeLa cells). The cytotoxic activities of C. procera may be due to the presence of various phytoconstituents such as cardenolides, flavonoids and other constituents 18-21. However, the molecular mechanism for such an effect needs further evaluation. Table 1: Percentage of inhibition of breast cancer (MCF-7) cells with respect to standard concentration of tamoxifen 4 1.445 97.18 2.82 8 1.361 91.53 8.47 16 1.149 77.27 22.73 32 1.106 74.38 25.62 64 1.093 73.50 26.50 128 0.976 65.64 34.36 256 0.627 42.17 57.83 512 0.588 39.54 60.46 % Inhibition = 100%viability Table 2: Percentage of inhibition of breast cancer (MCF-7) cells with respect to test sample: 1(DL extract) Conc. (μg/ml) Observed OD %Viability %Inhibition 4 1.361 91.53 8.47 8 1.287 86.55 13.45 16 1.202 80.83 19.17 32 1.165 78.35 21.65 64 1.035 69.60 30.40 128 0.987 66.38 33.62 256 0.942 63.35 35.65 512 0.802 53.93 46.07 % Inhibition = 100%viability Table 3: Percentage of inhibition of breast cancer (MCF-7) cells with respect to test sample: 2 (Flower extract) Conc. (μg/ml) Observed OD %Viability %Inhibition 4 1.305 87.76 12.24 8 1.211 81.44 18.56 16 1.184 79.62 20.38 32 1.133 76.19 23.81 64 0.933 66.78 33.22 128 0.961 64.63 35.37 256 0.912 61.33 38.67 512 0.838 56.36 43.64 67
Table 4: Percentage of inhibition of carcinoma of cervix (HeLa cells) with respect to standard concentration of tamoxifen 4 2.097 93.03 6.97 8 2.084 92.46 7.54 16 1.988 88.20 11.08 32 1.564 69.39 30.61 64 1.249 55.41 44.59 128 1.015 45.03 54.97 256 0.954 42.32 57.68 512 0.815 36.16 63.84 Table 5: Percentage of inhibition of carcinoma of cervix (HeLa cells) with respect to test sample: 1(DL extract) 4 2.152 95.47 4.53 8 1.956 86.78 13.22 16 1.889 83.81 16.19 32 1.755 77.86 22.14 64 1.602 71.07 28.93 128 1.445 64.11 35.89 256 1.369 60.74 39.26 512 1.265 56.12 43.88 Table 6: Percentage of inhibition of carcinoma of cervix (HeLa cells) with respect to test sample: 2 (Flower extract) 4 2.164 96.01 3.99 8 1.789 79.37 20.63 16 1.651 73.25 26.75 32 1.502 66.64 33.36 64 1.466 65.07 34.96 128 1.401 62.16 37.84 256 1.388 61.58 38.42 512 1.311 58.16 41.84 Fig. 1: Shows inhibition of breast cancer cells (MCF-7) by sample-1 and 2 with comparison to standard tamoxifen Fig. 2: Shows inhibition of carcinoma of cervix (HeLa cells) by sample-1 and 2 with comparison to standard tamoxifen 68
Fig. 3: Shows A) MCF-7 cells before treatment showing actively dividing cells, B) MCF-7 cells after treatment with sample-1 at 512μg/ml concentration incubated for 24hrs showing uncharacterized bodies, C) HeLa cells before treatment with samples or drug showing active cells grown in DMEM in a 96 well plate, D) HeLa cells after treatment with sample-1 at highest tested dose (512 μg/ml) showing unchracterized bodies. (All photographed viewed under inverted microscope) ACKNOWLEDGEMENT The authors are thankful to the management and principal of NRI College of Pharmacy, Pothavarappadu, Agiripalli mandal, Krishna Dist, A.P, India, for providing the research facilities and also to the Research Gateway for Biosciences, Dwarakanagar, Visakhapatnam, A.P. India, for carryingout the cell line culture work. REFERENCES 1. Kirtikar KR, Basu BD, Lolit Mohan Basu. Indian Medicinal Plants, Allahabad.1935: p. 1606. 2. Sharma GK. Calotropis procera and Calotropis gigantea. Indian Journal of Veterinary Sciences 1934; 4: 63 74. 3. Satyavati GV, Raina MK, Sharma M. Medicinal plants of India. Indian Council of Medical Research, New Delhi Vol. 1: 1976: p: 167 170. 4. Marcia Calheiros Chaves Silva, Antonio Batista da Silva, Fabiano Moura Teixeira, Paulo Cesar Pereira de Sousa, Regina Maria Macedo Rondon, Jose Eduardo Ribeiro Honorio Junior, et al. Therapeutic and biological activities of Calotropis procera (Ait.) R. Br.2010; 332-336. 5. Basu A, Nag Choudhuri AK. Preliminary studies on the antiinflammatory and analgesic activities of Calotropis procera root extract. Journal of Ethnopharmacology 1991; 31: 319 324. 6. Kumar VL, Basu N. Anti-inflammatory activity of the latex of Calotropis procera. Journal of Ethnopharmacology 1994; 44:123 125. 7. Dewan S, Sangraula H, Kumar VL. Preliminary studies on the analgesic activity of latex of Calotropis procera. Journal of Ethnopharmacology 2000; 73: 307 311. 8. Mueen Ahmed KK, Rana AC, Dixit VK.Free radical scavenging activity of Calotropis species. Indian Drugs 2003; 40: 654 655. 9. Mueen Ahmed KK, Rana AC, Dixit VK. Effect ofcalotropis procera latex on isoproterenol induced myocardial infarction in albino rats. Phytomedicine 2004; 11: 327 330. 10. Nandkarni, AK. Indian Materia Medica. 1: Popular Book Depot, Bombay.1976; p: 246. 11. De Oliveira JS, Bezerra DP, De Cdt, Filho Jdbm, De Moraces Mo, Pessoa C. In vitro cyto toxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R.Br. Toxicol in vitro 2007; 21:1563-1573. 12. Mosmann T.Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol Methods 1983; 65: 55. 13. Smit HF, Woerdenbag HJ, Singh RH, Meulenbeld GJ, Labadie RP and Zwaving JH. Ayurvedic herbal drugs with possible cytostatic activity. J Ethnopharmacol 1995; 47: 75 84. 14. Sehgal R, Roy S, and kumar VL. Evaluation of cytotoxic potential of latex of Calotropis procera and Podophyllotoxin in Allum cepa root model. Bio cell 2006: 30(1): 9-13. 15. Choedon T, Mathan G, Arya S, Kumar VL, Kumar V. Anticancer and cytotoxic properties of the Calotropis procera in a transgenic mouse model of hepatocellular carcinoma.world J Gastroenterol 2006;12: 2517-2522. 16. Oliveira JS, Bezerra DP, Freitas CDT, Filho JDBM, Moraes MO, Pessoa C, Costa-lotufo LV and Ramos MV. In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br. Toxicol in vitro 2007; 21: 1563 1573. 17. Choedon T, Mathan G, Arya S, Kumar VL, Kumar V. Anticancer and cytotoxic properties of the Calotropis procera in a transgenic mouse model of hepatocellular carcinoma. World J Gastroenterol 2006; 12: 2517-22. 69
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